Method of predicting genetic risk for hypertension

ABSTRACT

It is intended to provide a means of predicting genetic risk for hypertension at a high accuracy and high prediction possibility. Namely, risk for hypertension is predicted by a method involving the following steps: (i) the step of analyzing two or more polymorphisms selected from among 4 gene polymorphisms having been revealed as relating to hypertension; (ii) the step of determining the genotype of a nucleic acid sample based on the polymorphism data obtained in the above step; and (iii) the step of predicting the genetic risk for hypertension from the genotype thus determined.

TECHNICAL FIELD

The present invention relates to a detection method using genesassociated with hypertension. More particularly, it relates to adetection method using a plurality of gene polymorphisms associated withhypertension and to a kit used for the method. The present invention canbe used for diagnosing a risk of development of hypertension.

BACKGROUND ART

Hypertension is a complex multifactorial and polygenic disorder that isthought to result from an interaction between an individual's geneticbackground and various environmental factors (see non-patent document1). Given that hypertension is a major risk factor for coronary arterydisease, stroke, and chronic renal failure, prevention of hypertensionis an important public health goal. One approach to preventing thedevelopment of hypertension is to identify susceptibility genes. Linkagestudies (see non-patent documents 2 to 4) and association studies withcandictae genes (see non-patent documents 5 to 8) have implicatedvarious chromosomal loci and genes in predisposition to hypertension.Although genetic epidemiological studies have suggested that certaingenetic variants, including polymorphisms in the genes encodingangiotensinogen (non-patent document 5), α-adducin (non-patent document6), the β3 subunit of G proteins (non-patent document 7) and theβ2-adrenergic receptor (non-patent document 8), etc. increase the riskfor hypertension, the genes that contribute to genetic susceptibility tohypertension remain to be identified definitively. In addition, becauseof ethnic divergence of gene polymorphisms, it is important to constructa database of polymorphisms related to hypertension in each ethnicgroup.

Non-patent document 1: Lifton R P, Gharavi A G, Geller D S. Molecularmechanisms of human hypertension. Cell. 2001; 104: 545-556.

Non-patent document 2: Xu X, Rogus J J, Terwedow H A, Yang J, Wang Z,Chen C, Niu T, Wang B, Xu H, Weiss S, Schork N J, Fang Z. Anextreme-sib-pair genome scan for genes regulating blood pressure. Am JHum Genet. 1999; 64: 1694-1701.

Non-patent document 3: Krushkal J, Ferrell R, Mockrin S C, Turner S T,Sing C F, Boerwinkle E. Genome-wide linkage analysis of systolic bloodpressure using highly discordant siblings. Circulation. 1999; 99:1407-141.

Non-patent document 4: Rice T, Rankinen T, Province M A, Chagnon Y C,Perusse L, Borecki I B, Bouchard C, Rao D C. Genome-wide linkageanalysis of systolic and diastolic blood pressure: the Quebec FamilyStudy. Circulation. 2000; 102: 1956-1963.

Non-patent document 5: Jeunemaitre X, Soubrier F, Kotelevtsev Y V,Lifton R P, Williams C S, Charru A, Hunt S C, Hopkins P N, Williams R R,Laouel J-M, Corvol P. Molecular basis of human hypertension: role ofangiotensinogen. Cell. 1992; 71: 169-180.

Non-patent document 6: Cusi D, Barlassina C, Azzani T, Casari O,Citterio L, Devoto M, Gloriso N, Lanzani C, Manunta P, Righetti M,Rivera R, Stella P, Troffa C, Zagato L, Bianchi G. Polymorphisms ofa-adducin and salt sensitivity in patients with essential hypertension.Lancet. 1997; 349: 1353-1357.

Non-patent document 7: Siffert W, Rosskop D, Siffert G, Busch S, MoritzA, Erbel R, Sharma A M, Ritz E, Wichmann H-E, Jakobs K H, Horsthemke B.Association of a human G-protein β3 subunit variant with hypertension.Nat Genet. 1998; 18: 45-48.

Non-patent document 8: Bray M S, Krushkal J, Li L, Ferrell R, Kardia S,Sing C F, Turner S T, Boerwinkle E. Positional genomic analysisidentifies the 132-adrenergic receptor gene as a susceptibility locusfor hypertension. Circulation. 2000; 101: 2877-2882.

SUMMARY OF THE INVENTION

As mentioned above, many association studies have previously examinedthe relations between gene polymorphisms and hypertension. The resultsof most of these studies, however, remain controversial, with noconsensus on their implications, mainly because of the limitedpopulation size of the studies, the ethnic diversity of genepolymorphisms, and complicating environmental factors. Furthermore, eventhough associations with respect to hypertension have been detected, therelative risk (odds ratio) has tended to be low in large populations.

The present invention was made on the basis of the above-mentionedbackground, and the object thereof is to provide a means of diagnosinggenetic risk for hypertension with high accuracy and high predictabilityso as to contribute primary prevention of hypertension.

To achieve the above-mentioned objects, the present inventors haveextracted 71 genes which were estimated to be associated with coronaryarteriosclerosis, coronary artery spasm, hypertension, diabetesmellitus, hyperlipidemia, etc., and mainly selected 112 polymorphismswhich were predicted to be associated with functional changes of genesby the use of a plurality of public databases. Then, as to 112polymorphisms of 71 genes, association study with respect to myocardialinfarction was carried out in 445 myocardial cases and 464 controls. Asa result, the present inventors have identified 19 and 18 singlenucleotide polymorphisms (SNPs) related to myocardial infarction in menand women, respectively (Yamada Y, Izawa H, Ichihara S, et al.Prediction of the risk of myocardial infarction from polymorphisms incandidate genes. N Engl J Med. in press). However, these SNPs alsoinclude candidate determinants of the susceptibility to hypertension.Then, the present inventors therefore performed a large-scaleassociation study for these SNPs and hypertension. As a result, thepresent inventors succeeded in identifying four and four SNPs related tohypertension in men and women, respectively. In addition, analysis ofthe combination of these polymorphisms revealed maximal odds ratios of5.34 for men and 46.86 for women, respectively, on the basis of thestepwise forward selection method. In the analysis, the odds ratios weremaximum among the odds ratios which had been reported in the past. Basedon these results, it was possible to obtain findings that by selecting aplurality of SNPs from these SNPs and using the combination of theresults of analysis of each SNP, diagnosis of hypertension can becarried out with high reliability and high predictability. The presentinvention was made based on the above-mentioned findings and providesthe following configuration.

[1] A method for detecting the genotype in a nucleic acid sample, themethod comprising the following step (a):

(a) analyzing two or more polymorphisms selected from the groupconsisting of the following (1) to (4) in a nucleic acid sample:

(1) a polymorphism at the base number position 1648 of the glycoproteinIa gene;

(2) a polymorphism at the base number position 190 of the chemokinereceptor 2 gene;

(3) a polymorphism at the base number position 1100 of theapolipoprotein C-III gene; and

(4) a polymorphism at the base number position 825 of G-protein β3subunit gene.

[2] A method for detecting the genotype in a nucleic acid sample, themethod comprising the following step (b):

(b) analyzing two or more polymorphisms selected from the groupconsisting of the following (5) to (8) in a nucleic acid sample:

(5) a polymorphism at the base number position −850 of the tumornecrosis factor-α gene;

(6) a polymorphism at the base number position −238 of the tumornecrosis factor-α gene;

(7) a polymorphism at the base number position 3494 of the insulinreceptor substrate-1 gene; and

(8) a polymorphism at the base number position 1018 of the glycoproteinIba gene.

[3] A method for diagnosing the risk for hypertension, comprising thefollowing steps (i) to (iii):

(i) analyzing two or more polymorphisms selected from the groupconsisting of the following (1) to (4) in a nucleic acid sample:

(1) a polymorphism at the base number position 1648 of the glycoproteinIa gene;

(2) a polymorphism at the base number position 190 of the chemokinereceptor 2 gene;

(3) a polymorphism at the base number position 1100 of theapolipoprotein C-III gene; and

(4) a polymorphism at the base number position 825 of G-protein β3subunit gene.

(ii) determining, based on the information about polymorphism which wasobtained in the step (i), the genotype in the nucleic acid sample; and

(iii) assessing, based on the genotype determined, a genetic risk forhypertension.

[4] A method for diagnosing the risk for hypertension, comprising thefollowing steps (iv) to (vi):

(iv) analyzing two or more polymorphisms selected from the groupconsisting of the following (5) to (8) in a nucleic acid sample:

(5) a polymorphism at the base number position −850 of the tumornecrosis factor-α gene;

(6) a polymorphism at the base number position −238 of the tumornecrosis factor-α gene;

(7) a polymorphism at the base number position 3494 of the insulinreceptor substrate-1 gene; and

(8) a polymorphism at the base number position 1018 of the glycoproteinIba gene.

(v) determining, based on the information about polymorphism which wasobtained in the step (iv), the genotype in the nucleic acid sample; and

(vi) assessing, based on the genotype determined, a genetic risk forhypertension.

[5] A kit for detecting the genotype, comprising two or more of nucleicacids selected from the group consisting of the following (1) to (4):

(1) a nucleic acid for analyzing a polymorphism at the base numberposition 1648 of the glycoprotein Ia gene;

(2) a nucleic acid for analyzing a polymorphism at the base numberposition 190 of the chemokine receptor 2 gene;

(3) a nucleic acid for analyzing a polymorphism at the base numberposition 1100 of the apolipoprotein C-III gene; and

(4) a nucleic acid for analyzing a polymorphism at the base numberposition 825 of G-protein β3 subunit gene.

[6] A kit for detecting the genotype, comprising two or more of nucleicacids selected from the group consisting of the following (5) to (8):

(5) a nucleic acid for analyzing a polymorphism at the base numberposition −850 of the tumor necrosis factor-α gene;

(6) a nucleic acid for analyzing a polymorphism at the base numberposition −238 of the tumor necrosis factor-α gene;

(7) a nucleic acid for analyzing a polymorphism at the base numberposition 3494 of the insulin receptor substrate-1 gene; and

(8) a nucleic acid for analyzing a polymorphism at the base numberposition 1018 of the glycoprotein Iba gene.

[7] Fixed nucleic acids comprising the following two or more nucleicacids selected from the group consisting of the following (1) to (4)fixed to an insoluble support:

(1) a nucleic acid for analyzing a polymorphism at the base numberposition 1648 of the glycoprotein Ia gene;

(2) a nucleic acid for analyzing a polymorphism at the base numberposition 190 of the chemokine receptor 2 gene;

(3) a nucleic acid for analyzing a polymorphism at the base numberposition 1100 of the apolipoprotein C-III gene; and

(4) a nucleic acid for analyzing a polymorphism at the base numberposition 825 of G-protein β3 subunit gene.

[8] Fixed nucleic acids comprising the following two or more nucleicacids selected from the group consisting of the following (5) to (8)fixed to an insoluble support:

(5) a nucleic acid for analyzing a polymorphism at the base numberposition −850 of the tumor necrosis factor-α gene;

(6) a nucleic acid for analyzing a polymorphism at the base numberposition −238 of the tumor necrosis factor-α gene;

(7) a nucleic acid for analyzing a polymorphism at the base numberposition 3494 of the insulin receptor substrate-1 gene; and

(8) a nucleic acid for analyzing a polymorphism at the base numberposition 1018 of the glycoprotein Iba gene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a table summarizing 112 gene polymorphisms examined in ascreening association study in Examples.

FIG. 2 is also a table summarizing 112 gene polymorphisms examined in ascreening association study in Examples.

FIG. 3 is a table summarizing primers (SEQ ID NOs: 16, 17, 18, 14, 15,11, 12, 13, 8, 9, 10, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30in this order from the top), probes (SEQ ID NOs: 31, 32, 33 and 34 inthis order from the top) and other conditions used to determine thegenotype in Examples. In FIG. 3, FITC denotes fluoresceinisothiocyanate, TxR denotes Texas Red and Biotin denotes biotin,respectively.

FIG. 4 is a table summarizing single nucleotide polymorphisms examinedin an association study in Examples.

FIG. 5 is a table summarizing the background data of 1107 lesions in menand 833 lesions in women examined in an association study in Examples.Each data of age, body mass index, systolic blood pressure, diastolicblood pressure, and serum creatinine is represented by means±standarddeviation. In table, *1 denotes P<0.0001, *2 denotes P<0.001, and *3denotes P<0.01, respectively.

FIG. 6 is a table summarizing gene polymorphisms and results ofmultivariate logistic regression analysis examined in the associationstudy. In each SNP, smaller P value is expressed in boldface.

FIG. 7 is a view showing a distribution of genotype of genepolymorphisms associated with hypertension.

FIG. 8 is a table showing results of stepwise forward selection methodof multivariate logistic regression analysis of gene polymorphismsassociated with hypertension.

FIG. 9 is a table showing results of diagnosis of genetic risk forhypertension using a combination of four gene polymorphisms in men.

FIG. 10 is a table showing results of diagnosis of genetic risk forhypertension using a combination of four gene polymorphisms in women.

FIG. 11 is a graph showing a correlation between the cumulative oddsratio for hypertension and the number of single nucleotidepolymorphisms. (A) shows the correlation in men and (B) shows thecorrelation in women. In (A), SNPs include: SNP1: GPIa (1648A→G)polymorphism, SNP 2: CCR2 (190G→A) polymorphism, SNP3: ApoC-III(1100C→T) polymorphism, and SNP4: GPβ3 (825C→T) polymorphism. In (B),SNPs include: SNP1: TNFa (−850C→T) polymorphism, SNP2: TNFa (−238G→A)polymorphism, SNP3: IRS-1 (3494G→A) polymorphism, and SNP4: GPIba(1018C→T) polymorphism.

BEST MODE FOR CARRYING OUT THE INVENTION

The first aspect of the present invention relates to a method ofdetecting the genotype in a nucleic acid sample. One embodiment of thepresent invention is featured by including the step of analyzing two ormore polymorphisms selected from the group consisting of the following(1) to (4). Another embodiment is featured by including the step ofanalyzing two or more polymorphisms selected from the group consistingof the following (5) to (8). Note here that it is possible to determine,based on the information about polymorphisms which was obtained in theabove-mentioned step, the genotype in the nucleic acid sample, andthereby to assess, based on the genotype determined, a genetic risk forhypertension.

(1) a polymorphism at the base number position 1648 of the glycoproteinIa gene: 1648A→G (hereinafter, also referred to as “GPIa (1648A→G)polymorphism”)

(2) a polymorphism at the base number position 190 of the chemokinereceptor 2 gene: 190G→A (hereinafter, also referred to as “CCR2 (190G→A)polymorphism”)

(3) a polymorphism at the base number position 1100 of theapolipoprotein C-III gene: 1100C→T (hereinafter, also referred to as“ApoC-III (1100C→T) polymorphism”)

(4) a polymorphism at the base number position 825 of G-protein β3subunit gene: 825C→T (hereinafter, also referred to as “GPβ3 (825C→T)polymorphism”)

(5) a polymorphism at the base number position −850 of the tumornecrosis factor-α gene: −850C→T (hereinafter, also referred to as “TNFa(−850C→T) polymorphism”)

(6) a polymorphism at the base number position −238 of the tumornecrosis factor-α gene: −238G→A (hereinafter, also referred to as “TNFa(−238G→A) polymorphism”)

(7) a polymorphism at the base number position 3494 of the insulinreceptor substrate-1 gene: 3494G→A (hereinafter, also referred to as“IRS-1 (3494G→A) polymorphism”)

(8) a polymorphism at the base number position 1018 of the glycoproteinIba gene: 1018C→T (hereinafter, also referred to as “GPIba (1018C→T)polymorphism”)

In the above, description such as 1648A→G means that polymorphism at therelevant base number position consists of two genotypes, bases beforeand after the arrow.

The base number of each gene is expressed using as standards the knownsequences which are registered in the public database, GenBank (NCBI).Note here that in the base sequence of SEQ ID NO: 1 (Accession No.X17033 M28249: Human mRNA for integrin alpha-2 subunit), the 1648th basecorresponds to the base at position 1648 of the glycoprotein Ia gene.Similarly, in the base sequence of SEQ ID NO: 2 (Accession No. U95626:Homo sapiens ccr2b (ccr2), ccr2a (ccr2), ccr5 (ccr5) and ccr6(ccr6)genes, complete cds, and lactoferrin (lactoferrin) gene, partial cds,complete sequence (wherein, sequence of SEQ ID NO: 2 is a sequence to50,000th base sequence)), the 46295th base corresponds to the base atposition 190 of the chemokine receptor 2 gene; in the base sequence ofSEQ ID NO: 3 (Accession No. X01392: Human apolipoprotein CIII gene andapo AI-apo CIII intergenic), the 1100th base corresponds to the base atposition 1100 of the apolipoprotein C-III gene; in the base sequence ofSEQ ID NO: 4 (Accession No. M31328: Human guanine nucleotide-bindingprotein beta-3 subunit mRNA, complete cds.), the 831st base correspondsto the base at position 825 of the G-protein β3 subunit gene; in thebase sequence of SEQ ID NO: 5 (Accession No. L11698: Homo sapiens tumornecrosis factor alpha gene, promoter region.), the 203rd basecorresponds to the base at position −850 of the tumor necrosis factor agene; in the sequence of SEQ ID NO: 5 (Accession No. L11698: Homosapiens tumor necrosis factor alpha gene, promoter region.), the 816thbase corresponds to the base at position −238 of the tumor necrosisfactor a gene; in the sequence of SEQ ID NO: 6 (Accession No. S85963:hIRS-1=rat insulin receptor substrate-1 homolog [human, cell line FOCUS,Genomic, 6152 nt]), the 3494th base corresponds to the base at position3494 of the insulin receptor substrate-1 gene; and in the sequence ofSEQ ID NO: 7 (Accession No. J02940: Human platelet glycoprotein Ib alphachain mRNA, complete cds.), the 524th base corresponds to the base atposition 1018 of the glycoprotein Iba gene.

In the present invention, “analyzing polymorphism” means theinvestigation as to what genotype a nucleic acid sample has in the genepolymorphism to be analyzed. It is the same meaning as the investigationon the base (base sequence) of the position in which the polymorphismexists. Typically, for example, in the case of the analysis of the GPIa(1648A→G) polymorphism, it refers to investigation on what genotype,i.e., AA (the base at position 1648 is a homozygote of allele A), AG(the base at position 1648 is a heterozygote of allele A and allele G)and GG (the base at position 1648 is a homozygote of allele G), theglycoprotein Ia gene in a nucleic acid sample has.

As shown in Examples mentioned below, the polymorphisms mentioned (1) to(4) above are polymorphisms that are recognized as being particularlyeffective to be used in determining genetic risk for hypertension in ananalysis of Japanese male subjects. Therefore, analysis targeting thesepolymorphisms enables diagnosis with higher accuracy and with higherpredictability when subjects are men (particularly, Japanese men).

Similarly, as shown in Examples mentioned below, the polymorphismsmentioned (5) to (8) above are polymorphisms that are recognized asbeing particularly effective to be used in determining genetic risk forhypertension in an analysis of Japanese female subjects. Therefore,analysis targeting these polymorphisms enables diagnosis with higheraccuracy and with higher predictability when subjects are women(particularly, Japanese women).

Herein, in principle, in proportion to the increase in the number ofpolymorphisms to be analyzed, the genotypes of nucleic acid sample areclassified more finely. Thereby, it is possible to diagnose a geneticrisk for hypertension with higher predictability. From this viewpoint,it is preferable to detect the genotype by analyzing a larger number ofpolymorphisms in the above-mentioned polymorphisms (1) to (4).Therefore, it is the most preferable to analyze all of the polymorphisms(1) to (4). In the case where detection is carried out by combiningthree or less of polymorphisms, it is preferable to preferentiallyselect the polymorphisms with higher odds ratios as in Examplesmentioned below. For example, in the case where three polymorphisms areused in combination, it is preferable to select three polymorphisms withhigher odds ratio, that is, to select (2), (3) and (4). Similarly, inthe case where two polymorphisms are used in combination, it ispreferable to select (2) and (4).

Similarly, in the case where two or more polymorphisms selected from thegroup consisting of polymorphisms (5) to (8), it is most preferable toanalyze all these polymorphisms, that is, four polymorphisms. In thecase where detection is carried out by combining three or less ofpolymorphisms, it is preferable to preferentially select thepolymorphisms with higher odds ratios in Examples mentioned below. Forexample, in the case where three polymorphisms are used in combination,it is preferable to select (5), (7) and (8). Similarly, in the casewhere two polymorphisms are used in combination, it is preferable toselect (5) and (7).

A method for analyzing each genetic polymorphism is not particularlylimited and known method can be employed. The known methods may include,for example, amplification by PCR using an allele-specific primer (andprobe), a method for analyzing polymorphism of amplified product bymeans of fluorescence or luminescence; a method using a PCR (polymerasechain reaction) method including a PCR-RFLP (polymerase chainreaction-restriction fragment length polymorphism) method, a PCR-SSCP(polymerase chain reaction-single strand conformation polymorphism)method (Orita, M. et al., Proc. Natl. Acad. Sci., U.S.A., 86, 2766-2770(1989), etc.), and a PCR-SSO (specific sequence oligonucleotide) method,an ASO (allele specific oligonucleotide) hybridization method combiningthe PCR-SSO method and a dot hybridization method (Saiki, Nature, 324,163-166 (1986), etc.), or a TaqMan-PCR method (Livak, K J, Genet Anal,14, 143 (1999), Morris, T. et al., J. Clin. Microbiol., 34, 2933(1996)), an Invader method (Lyamichev V et al., Nat Biotechnol, 17, 292(1999)), a MALDI-TOF/MS (matrix) method using a primer extension method(Haff L A, Smirnov I P, Genome Res 7, 378 (1997)), a RCA (rolling cycleamplification) method (Lizardi P M et al., Nat Genet 19, 225 (1998)), amethod using DNA microchip or micro-array (Wang D G et al., Science 280,1077 (1998), etc.)), a primer extension method, a Southern blothybridization method, a dot hybridization method (Southern, E., J. Mol.Biol. 98, 503-517 (1975)), etc.), or the like. Furthermore, an analysismay be made by direct sequencing of the portion of polymorphism which issubject to analysis. Note here that polymorphisms may be analyzed bycombining these methods ad libitum.

In the case where the amount of nucleic acid sample is small, it ispreferable to analyze it by a method using PCR (for example, PCR-RFLPmethod) from the viewpoint of detection sensitivity or accuracy.Furthermore, any of the above-mentioned analysis methods may be employedafter nucleic acid sample is amplified in advance (including a partialregion of nucleic acid sample) by a gene amplification such as PCRmethod or a method applying PCR method.

Meanwhile, in the case where a large number of nucleic acid samples areanalyzed, it is particularly preferable to employ a method capable ofanalyzing a large number of samples in a relatively short period oftime, for example, allele-specific PCR method, allele-specifichybridization method, TaqMan-PCR method, Invader method, MALDI-TOF/MS(matrix) method using a primary extension method, RCA (rolling cycleamplification) method, a method using a DNA chip or a micro-array, orthe like.

The above methods use nucleic acids (also called “nucleic acid foranalyzing polymorphism” in the present invention), e.g., primer andprobe, in accordance with each method. An example of the nucleic acidsfor analyzing polymorphism may include a nucleic acid having a sequencecomplementary to a given region including the site of polymorphism(partial DNA region) in a gene which contains polymorphism to beanalyzed, and a nucleic acid (primer) which has a sequence complementaryto a given region including the site of polymorphism (partial DNAregion) in a gene which contains polymorphism to be analyzed and whichis designed to specifically amplify the DNA fragment containing therelevant site of polymorphism. In the case where polymorphism atposition 1648 of the glycoprotein Ia gene is a subject to be analyzed,an example of such nucleic acids includes a nucleic acid having asequence complementary to a partial DNA region including the position1648 of the glycoprotein Ia gene whose base at position 1648 is A(adenine), or a nucleic acid having a sequence complementary to apartial DNA region including the position 1648 of the glycoprotein Iagene whose base at position 1648 is G (guanine).

Other concrete examples of nucleic acids for analyzing polymorphism mayinclude a set of nucleic acids which is designed to specifically amplifythe partial DNA region that contains the relevant site of polymorphismonly in the case where the site of polymorphism to be analyzed is acertain genotype. A more concrete example may include a set of nucleicacids which is designed to specifically amplify the partial DNA regionincluding the site of polymorphism to be analyzed and which consists ofa sense primer that specifically hybridizes the partial DNA regionincluding the relevant site of polymorphism in an antisense strand whosesite of polymorphism is a certain genotype and of an antisense primerthat specifically hybridizes a partial region in the sense strand. Inthe case where a subject to the analysis is a polymorphism at position1648 of the glycoprotein Ia gene, examples of such a set of nucleicacids include a set of nucleic acids which is designed to specificallyamplify the partial DNA region including the base at position 1648 ofthe glycoprotein Ia gene and which consists of a sense primer thatspecifically hybridizes the partial DNA region containing the base atposition 1648 in the antisense strand of the glycoprotein Ia gene whosebase at position 1648 is A (adenine) and of an antisense primer thatspecifically hybridizes a partial region in the sense strand; or a setof nucleic acids which consists of a sense primer that specificallyhybridizes the partial DNA region including the base at position 1648 inthe antisense strand of the glycoprotein Ia gene whose base at position1648 is G (guanine) and of an antisense primer that specificallyhybridizes a partial region in the sense strand. The length of thepartial DNA region to be amplified here is set accordingly in a rangewhich is appropriate for its detection, and is for example, 50 bp to 200bp, and preferably 80 bp to 150 bp. A more concrete example may includea set of nucleic acid primers for analyzing the GPIa (1648A→G)polymorphism containing the following sequences. Note here that anunderlined part in the following sequences means a part corresponding tothe polymorphism. Furthermore, in the sequence, N denotes any of A, T, Cand G. sense primer GAGTCTACCTGTTTACTATCAANAA:, SEQ ID NO: 8 orGAGTCTACCTGTTTACTATCAANGA: SEQ ID NO: 9 antisense primerACCAGTACTAAAGCAAATTAAACT: SEQ ID NO: 10

Similarly, an example of a nucleic acid primer for analyzing the CCR2(190G→A) polymorphism may include a set containing the followingsequences. antisense primer GCAGTTTATTAAGATGAGGNCG:, SEQ ID NO: 11 orTTGCAGTTTATTAAGATGAGGNTG: SEQ ID NO: 12 sense primerGGTGCTCCCTGTCATAAATTTGA: SEQ ID NO: 13

Similarly, an example of a nucleic acid primer for analyzing theApoC-III (1100C→T) polymorphism may include a set containing thefollowing sequences. sense primer CCTTCTCAGCTTCATGCAGG:, SEQ ID NO: 14antisense primer GTCTTGGTGGCGTGCTTCA: SEQ ID NO: 15

Similarly, an example of a nucleic acid primer for analyzing the GPβ3(825C→T) polymorphism may include a set containing the followingsequences. sense primer TCTGCGGCATCACGTNCG:, SEQ ID NO: 16 orTCTGCGGCATCACGTNTG: SEQ ID NO: 17 antisense primer GAATAGTAGGCGGCCACTGA:SEQ ID NO: 18

Similarly, an example of a nucleic acid primer for analyzing the TNFa(−850C→T) polymorphism may include a set containing the followingsequences. antisense primer TCTACATGGCCCTGTCTTNGT:, SEQ ID NO: 19 orCTCTACATGGCCCTGTCTTNAT: SEQ ID NO: 20 sense primerCTCTACATGGCCCTGTCTTTAT: SEQ ID NO: 21

Similarly, an example of a nucleic acid primer for analyzing the TNFa(−238G→A) polymorphism may include a set containing the followingsequences. antisense primer CCCCATCCTCCCTGCTNCG:, SEQ ID NO: 22 orCCCCATCCTCCCTGCTNTG: SEQ ID NO: 23 sense primer AGTCAGTGGCCCAGAAGACC:SEQ ID NO: 24

Similarly, an example of a nucleic acid primer for analyzing the IRS-1(3494G→A) polymorphism may include a set containing the followingsequences. sense primer GGGCCCTGCACCTCCNGG:, SEQ ID NO: 25 orGGGCCCTGCACCTCCNAG: SEQ ID NO: 26 antisense primer GGGTAGGCCTGCAAATGCTA:SEQ ID NO: 27

Similarly, an example of a nucleic acid primer for analyzing the GPIba(1018C→T) polymorphism may include a set containing the followingsequences. sense primer CCCAGGGCTCCTGNCG:, SEQ ID NO: 28 orCCCCAGGGCTCCTGNTG: SEQ ID NO: 29 antisense primer TGAGCTTCTCCAGCTTGGGTG:SEQ ID NO: 30

On the other hand, a concrete example of the probe can include:

-   -   as a probe for analyzing ApoC-III (1100C→T) polymorphism,    -   CAGCTTCATGCAGGGCTACA: SEQ ID NO: 31, or    -   CAGCTTCATGCAGGGTTACA: SEQ ID NO: 32    -   as a probe for analyzing TNFa (−850C→T) polymorphism,    -   ACATGGCCCTGTCTTNGTTAAG: SEQ ID NO: 33, or    -   ACATGGCCCTGTCTTNATTAAG: SEQ ID NO: 34    -   as a probe for analyzing IRS-1 (3494G→A) polymorphism,    -   CACCTCCNGGGGCTGCTAG: SEQ ID NO: 35, or    -   CACCTCCNAGGGCTGCTAG: SEQ ID NO: 36

The above nucleic acid primers and nucleic acid probes are mereexamples. Nucleic acid primers may contain a partially modified basesequence as long as they can carry out the aimed amplification reactionwithout inconvenience, while nucleic acid probes may contain a partiallymodified base sequence as long as they can carry out the aimedhybridization reaction without inconvenience. “Partially modified”herein means that a part of bases is deleted, replaced, inserted, and/oradded. The numbers of bases to be modified are, for example, one toseven, preferably one to five, and more preferably one to three. Notehere that such a modification is made in the portions other than basescorresponding to the site of polymorphism, in principle.

As nucleic acids (probes or primers) for analyzing polymorphism, DNAfragments or RNA fragments are used accordingly in response to theanalysis method employed. The base length of nucleic acids for analyzingpolymorphism may be sufficient if it exerts respective functions of eachnucleic acid. Base lengths in the case of use as primers are, forexample, 10 bp to 50 bp, preferably 15 bp to 40 bp, and more preferably15 bp to 30 bp.

Note here that in the case of use as primers, some mismatches to thesequence which constitutes the template may be admitted as long as theprimer can specifically hybridize the subject for amplification andamplify the target DNA fragment. In the case of probes, some mismatchesto the sequence which is subject to detection may be similarly admittedas long as the probe can specifically hybridize the sequence which issubject to detection. The numbers of mismatches are one to several,preferably one to five, and more preferably one to three.

Nucleic acids (primers and probes) for analyzing polymorphism can besynthesized in accordance with known methods, e.g., phosphodiestermethod. Note here that textbooks (e.g., Molecular Cloning, ThirdEdition, Cold Spring Harbor Laboratory Press, New York) can be referredwith respect to design, synthesis, and others of nucleic acids foranalyzing polymorphism.

Nucleic acids for analyzing polymorphism in the present invention can belabeled with labeling substances in advance. The use of such labelednucleic acids allows, for example, the analysis of polymorphism by usingthe labeling amount in the product of amplification as a marker.Furthermore, by labeling two kinds of primers which were designedspecifically amplify the partial DNA region in the gene of each genotypethat constitute polymorphism with labeling substances that are differentfrom each other, the genotype in a nucleic acid sample can bediscriminated according to the labeling substance and labeling amount tobe detected based on the product of amplification. Concrete examples ofdetection methods using these labeled primers may include: a method fordetecting polymorphism, which includes labeling, with fluoresceinisothiocyanate and Texas red, two kinds of nucleic acid primers(allele-specific sense primers) that respectively and specificallyhybridize the sense strand of each genotype constituting polymorphism;amplifying the partial DNA region including the site of polymorphism byusing these labeled primers and the antisense primers that specificallyhybridize the antisense strand; and measuring the labeling amount ofeach fluorescent substance in the product of amplification obtained.Note here that labeling of the antisense primer herein with, forexample, biotin allows the separation of the product of amplification byutilizing the specific binding between biotin and avidin.

Radioactive isotopes, for example, ³²P, and fluorescent substance, forexample, fluorescein isothiocyanate, tetramethylrhodamineisothiocyanate, and Texas red, etc. can be exemplified as labelingsubstances to be used in labeling nucleic acids for analyzingpolymorphism. The 5′ terminal labeling method using alkaline phosphataseand T4 polynucleotide kinase, the 3′ terminal labeling method using T4DNA polymerase and Klenow fragment, nicktranslation method, randomprimer method (Molecular Cloning, Third Edition, Chapter 9, Cold SpringHarbor Laboratory Press, New York), and the like can be exemplified aslabeling methods.

The above-mentioned nucleic acids for analyzing polymorphism can be usedalso under a condition fixed to an insoluble support. Processing of aninsoluble support to be used for the fixation to several forms such aschips and beads allows the more simplified analysis of polymorphism byusing these fixed nucleic acids.

A nucleic acid sample can be prepared from blood, skin cells, mucouscells, hair, and others from the subject according to known extractionmethods and purification methods. In the case of including the genewhich is subject to the analysis of polymorphism, the genome DNA ofarbitrary length can be used as a nucleic acid sample. Furthermore, itis not necessary to use a nucleic acid sample in which all genes subjectto the analysis are present on one nucleic acid. That is to say, as anucleic acid sample of the present invention, both material in which allgenes subject to the analysis are present on one nucleic acid andmaterial in which genes subject to the analysis are present separatelyon two or more nucleic acids can be used. Note here that material in afragmented or partial condition may be accepted as long as the site ofpolymorphism to be analyzed is at least present, although genes subjectto the analysis in a nucleic acid sample are not in a complete condition(i.e., a condition in which the full length of the gene is present).

Analysis of each gene polymorphism is carried out each by each of thegene polymorphism, or a plurality or entire gene polymorphisms arecarried out simultaneously. In the former case, for example, nucleicacid sample collected from the subjects is divided in accordance withthe number of polymorphisms to be analyzed, and analysis of polymorphismis carried out individually. In the latter case, for example, analysisof polymorphism can be carried out by DNA chip or micro-array. Note herethat “simultaneously” herein not only imply that all operations of theanalysis process are conducted simultaneously but also include the casein which part of operations (e.g., operation to amplify nucleic acid,hybridization or detection of the probe) is conducted simultaneously.

Polymorphism of each gene can be analyzed by using mRNA which is aproduct of transcription of the gene which is subject to the analysis.After extracting and purifying mRNA of the gene, which is subject to theanalysis, from blood, urine, and others of the subject, for example,polymorphism can be analyzed with mRNA as a starting material byconducting methods, e.g., Northern blotting method (Molecular Cloning,Third Edition, 7.42, Cold Spring Harbor Laboratory Press, New York), dotblotting method (Molecular Cloning, Third Edition, 7.46, Cold SpringHarbor Laboratory Press, New York), RT-PCR method (Molecular Cloning,Third Edition, 8.46, Cold Spring Harbor Laboratory Press, New York), andmethods using the DNA chip (DNA array), and the like.

In addition, in the above-mentioned polymorphism, polymorphism involvedwith changes in amino acids can be analyzed by using the expressionproduct of gene that is a subject to analysis. In this case, material,even if it is partial protein or partial peptide, can be used as asample for analysis as long as it contains amino acids which correspondto the site of polymorphism.

Analysis methods using these expression products of gene may include: amethod for directly analyzing amino acids at the site of polymorphism, amethod for immunologically analyzing utilizing changes ofthree-dimensional structure, or the like. As the former method, awell-known amino acid sequence analysis method (a method using Edmanmethod) can be used. As the latter method, ELISA (enzyme-linkedimmunosorbent assay) using a monoclonal antibody or polyclonal antibodywhich has binding activity specific to the expression product of genewhich has any of genotypes that constitute polymorphism;radioimmunoassay, immunoprecipitation method, immunodiffusion method,and the like, can be used.

Information about polymorphisms to be obtained by conducting thedetection methods of the present invention described above can be usedto diagnose a genetic risk for hypertension. That is to say, the presentinvention also provides a method for diagnosing a genetic risk forhypertension, which includes a step of determining the genotype in anucleic acid sample based on information about polymorphisms obtained bythe above-detection methods, and a step of assessing a genetic risk forhypertension based on the determined genotype in the nucleic acidsample. Herein, the determination of the genotype is typically todetermine which genotype both alleles of nucleic acid samples have withrespect to the polymorphism to be detected. In the case where thesubject to be detected is, for example, GPIa (1648A→G) polymorphism, thedetection of genotype is typically, an investigation on what genotypefrom AA (the base at position 1648 is a homozygote of allele A), AG (thebase at position 1648 is a heterozygote of allele A and allele G) and GG(the base at position 1648 is a homozygote of allele G), the GPIa genehas in a nucleic acid sample.

By considering the results obtained in Example mentioned below, in orderto enable a diagnosis of genetic risk for hypertension with highaccuracy and high predictability, for example, in the case of the GPIa(1648A→G) polymorphism, it is determined whether the genotype in anucleic acid sample is GG or, AA or AG. Similarly, in the case of theCCR2 (190G→A) polymorphism, it is determined whether the genotype is AA,or GG or GA; in the case of the ApoC-III (1100C→T) polymorphism, it isdetermined whether the genotype is TT, or CC or CT; in the case of theGPβ3 (825C→T) polymorphism, it is determined whether the genotype is CTor TT, or CC; in the case of the TNFa (−850C→T) polymorphism, it isdetermined whether the genotype is TT, or CC or CT; in the case of TNFa(−238G→A) polymorphism, it is determined whether the genotype is GA orAA, or GG; in the case of IRS-1 (3494G→A) polymorphism, it is determinedwhether the genotype is GA or AA, or GG; and in the case of GPIba(1018C→T) polymorphism, it is determined whether the genotype is CT orTT, or CC.

Diagnosis of a genetic risk for hypertension enables prediction ofpotentiality (likelihood of development) in that hypertension might bedeveloped in future, that it to say, development risk (susceptibility todevelopment). Furthermore, it becomes possible to carry out therecognition of hypertension based on the genotype that is an objectiveindex or to understand conditions of the disease. In other words, thediagnosis method of the present invention makes it possible to evaluatethe risk of development of hypertension, to recognize the development ofhypertension, or to understand conditions of the disease. It isclinically significant that it is possible to assess the risk ofdevelopment because having knowledge about the development risk inadvance contributes to primary prevention of hypertension so as to makesit possible to take an appropriate prevention.

The information obtained by the diagnosis method of the presentinvention can be used for selecting an appropriate treatment,improvement of prognosis, improvement of QOL (quality of life) ofpatients, reduction of the risk of development, or the like.

By conducting the diagnosis method of the present invention, it ispossible to monitor the development risk for hypertension, etc. As aresult of such monitoring, when correlation between certain externalfactors (environment factor, administration of drugs, and the like) andthe increase in the risk of development is found, the relevant externalfactors are recognized as risk factors and it can be thought that basedon such information, the development risk etc. can be reduced.

The genetic information associated with the development of hypertensionobtained by the present invention can be used for treatment ofhypertension (including preventive treatment). For example, as a resultof carrying out the diagnostic method of the present invention, when thepolymorphism to be analyzed is a genotype to increase the risk ofdevelopment of hypertension, by introducing a gene having a genotypewith low risk of development into a living body and allowing the gene toexpress, the reduction of disease, suppression of development andreduction of development risk, and the like can be expected due to theexpression of the gene. The same treatment effect can be expected by amethod of introducing antisense strand with respect to mRNA of genehaving a genotype with high risk of development and suppressing theexpression of the mRNA.

The introduction of genes or antisense strand can be carried out by amethod, for example, a method using a plasmid for gene introduction or avirus vector, electroporation (Potter, H. et al., Proc. Natl. Acad. Sci.U.S.A. 81, 7161-7165(1984), an ultrasonic micro bubble (Lawrie, A., etal. Gene Therapy 7, 2023-2027 (2000)), lipofection (Felgner, P. L. etal., Proc. Natl. Acad. Sci. U.S.A. 84, 7413-7417 (1984)), microinjection(Graessmann, M. & Graessmann, A., Proc. Natl. Acad. Sci. U.S.A.73,366-370(1976)), and the like. By utilizing these methods, desiredgenes, etc. can be directly introduced (in vivo method) or indirectlyintroduced (ex vivo method).

The second aspect of the present invention provides a kit (a kit fordetecting the genotype or a kit for diagnosing hypertension) to be usedin the above-mentioned detecting method or diagnostic method in thepresent invention. Such a kit contains nucleic acids (nucleic acids foranalyzing polymorphism) for analyzing two or more polymorphisms selectedfrom the group consisting of polymorphisms described in (1) to (4)above. As another embodiment, such a kit is constructed, which containsnucleic acids (nucleic acids for analyzing polymorphism) for analyzingtwo or more polymorphisms selected from the group consisting ofpolymorphisms described in (5) to (8) above.

Nucleic acids for analyzing polymorphism are designed as materials whichcan specifically amplifies (primer) or specifically detect (probe) theDNA region containing the polymorphism portion to be analyzed or mRNAwhich corresponds to the region in the analysis methods to be applied (amethod which utilizes PCR using the above-mentioned allele-specificnucleic acids and the like, PCR-RFLP method, PCR-SSCP method, TaqMan-PCRmethod, Invader method, etc.). Concrete examples of kits to be providedaccording to the present invention are described below.

A kit for detecting the genotype, comprising two or more nucleic acidsselected from the group consisting of the following (1) to (4):

(1) a nucleic acid having a sequence which is complementary to thepartial DNA region containing the base at position 1648 of theglycoprotein Ia gene whose base at position 1648 is A, or a nucleic acidhaving a sequence which is complementary to the partial DNA regioncontaining the base at position 1648 of the glycoprotein Ia gene whosebase at position 1648 is G;

(2) a nucleic acid having a sequence which is complementary to thepartial DNA region containing the base at position 190 of the chemokinereceptor 2 gene whose base at position 190 is G, or a nucleic acidhaving a sequence which is complementary to the partial DNA regioncontaining the base at position 190 of the chemokine receptor 2 genewhose base at position 190 is A;

(3) a nucleic acid having a sequence which is complementary to thepartial DNA region containing the base at position 1100 of theapolipoproteins C-III gene whose base at position 1100 is C, or anucleic acid having a sequence which is complementary to the partial DNAregion containing the base at position 1100 of the apolipoproteins C-IIIgene whose base at position 1100 is T; and

(4) a nucleic acid having a sequence which is complementary to thepartial DNA region containing the base at position 825 of the G-proteinβ3 subunit gene whose base at position 825 is C, or a nucleic acidhaving a sequence which is complementary to the partial DNA regioncontaining the base at position −482 of the G-protein β3 subunit genewhose base at position 825 is T.

In the above mention, kits are constructed by selecting two or morenucleic acids from the group consisting of (1) to (4). However, kits maybe constructed by making a group consisting of two or more nucleic acidsarbitrarily selected from (1) to (4) and selecting two or more nucleicacids from such a group. For example, kits may be constructed byselecting two or more nucleic acids from the group consisting of (2) to(4) (nucleic acids for analyzing polymorphisms with three highest oddsratio).

A kit for detecting the genotype, comprising two or more nucleic acidsselected from the group consisting of the following (5) to (8):

(5) a nucleic acid having a sequence which is complementary to thepartial DNA region containing the base at position −850 of the tumornecrosis factor-α gene whose base at position −850 is C, or a nucleicacid having a sequence which is complementary to the partial DNA regioncontaining the base at position −850 of the tumor necrosis factor-α genewhose base at position −850 is T;

(6) a nucleic acid having a sequence which is complementary to thepartial DNA region containing the base at position −238 of the tumornecrosis factor-α gene whose base at position −238 is Q or a nucleicacid having a sequence which is complementary to the partial DNA regioncontaining the base at position −238 of the tumor necrosis factor-α genewhose base at position −238 is A;

(7) a nucleic acid having a sequence which is complementary to thepartial DNA region containing the base at position 3494 of the insulinreceptor substrate-1 gene whose base at position 3494 is G, or a nucleicacid having a sequence which is complementary to the partial DNA regioncontaining the base at position 3494 of the insulin receptor substrate-1gene whose base at position 3494 is A; and

(8) a nucleic acid having a sequence which is complementary to thepartial DNA region containing the base at position 1018 of theglycoprotein Iba gene whose base at position 1018 is C, or a nucleicacid having a sequence which is complementary to the partial DNA regioncontaining the base at position 1018 of the glycoprotein Iba gene whosebase at position 1018 is T.

In the above mention, kits are constructed by selecting two or morenucleic acids from the group consisting of (5) to (8). However, kits maybe constructed by making a group consisting of two or more nucleic acidsarbitrarily selected from (5) to (8) and selecting two or more nucleicacids from such a group. For example, kits may be constructed byselecting two or more nucleic acids from the group consisting of (5),(7) and (8) (nucleic acids for analyzing polymorphisms with threehighest odds ratios in Example mentioned below).

A kit for detecting the genotype, comprising two or more sets of nucleicacids selected from the group consisting of the following (1) to (4):

(1) a set of nucleic acids which is designed to specifically amplify thepartial DNA region containing the base at position 1648 of theglycoprotein Ia gene only in the case where the base at position 1648 ofthe glycoprotein Ia gene in a nucleic acid sample is A, or a set ofnucleic acids which is designed to specifically amplify the partial DNAregion containing the base at position 1648 of the glycoprotein Ia geneonly in the case where the base at position 1648 of the glycoprotein Iagene in a nucleic acid sample is G;

(2) a set of nucleic acids which is designed to specifically amplify thepartial DNA region containing the base at position 190 of the chemokinereceptor 2 gene only in the case where the base at position 190 of thechemokine receptor 2 gene in a nucleic acid sample is G, or a set ofnucleic acids which is designed to specifically amplify the partial DNAregion containing the base at position 190 of the chemokine receptor 2gene only in the case where the base at position 190 of the chemokinereceptor 2 gene in a nucleic acid sample is A;

(3) a set of nucleic acids which is designed to specifically amplify thepartial DNA region containing the base at position 1100 of theapolipoprotein C-III gene only in the case where the base at position1100 of the apolipoprotein C-III gene in a nucleic acid sample is C, ora set of nucleic acids which is designed to specifically amplify thepartial DNA region containing the base at position 1100 of theapolipoprotein C-III gene only in the case where the base at position1100 of the apolipoprotein C-III gene in a nucleic acid sample is T; and

(4) a set of nucleic acids which is designed to specifically amplify thepartial DNA region containing the base at position 825 of the G-proteinβ3 subunit gene only in the case where the base at position 825 of theG-protein β3 subunit gene in a nucleic acid sample is C, or a set ofnucleic acids which is designed to specifically amplify the partial DNAregion containing the base at position 825 of the G-protein β3 subunitgene only in the case where the base at position 825 of the G-protein β3subunit gene in a nucleic acid sample is T.

In the above mention, kits are constructed by selecting two or morenucleic acids from the group consisting of (1) to (4). However, kits maybe constructed by making a group consisting of two or more nucleic acidsarbitrarily selected from (1) to (4) and selecting two or more nucleicacids from such a group. For example, kits may be constructed byselecting two or more nucleic acids from the group consisting of (2) to(4) (nucleic acids for analyzing polymorphisms with three highest oddsratio in Example mentioned below).

A kit for detecting the genotype, comprising two or more sets of nucleicacids selected from the group consisting of the following (5) to (8):

(5) a set of nucleic acids which is designed to specifically amplify thepartial DNA region containing the base at position −850 of the tumornecrosis factor-α gene only in the case where the base at position −850of the tumor necrosis factor-α gene in a nucleic acid sample is C, or aset of nucleic acids which is designed to specifically amplify thepartial DNA region containing the base at position −850 of the tumornecrosis factor-α gene only in the case where the base at position −850of the tumor necrosis factor-α gene in a nucleic acid sample is T;

(6) a set of nucleic acids which is designed to specifically amplify thepartial DNA region containing the base at position −238 of the tumornecrosis factor-α gene only in the case where the base at position −238of the tumor necrosis factor-α gene in a nucleic acid sample is G, or aset of nucleic acids which is designed to specifically amplify thepartial DNA region containing the base at position −238 of the tumornecrosis factor-α gene only in the case where the base at position −238of the tumor necrosis factor-α gene in a nucleic acid sample is A;

(7) a set of nucleic acids which is designed to specifically amplify thepartial DNA region containing the base at position 3494 of the insulinreceptor substrate-1 gene only in the case where the base at position3494 of the insulin receptor substrate-1 gene in a nucleic acid sampleis G, or a set of nucleic acids which is designed to specificallyamplify the partial DNA region containing the base at position 3494 ofthe insulin receptor substrate-1 gene only in the case where the base atposition 3494 of the insulin receptor substrate-1 gene in a nucleic acidsample is A; and

(8) a set of nucleic acids which is designed to specifically amplify thepartial DNA region containing the base at position 1018 of theglycoprotein Iba gene only in the case where the base at position 1018of the glycoprotein Iba gene in a nucleic acid sample is C, or a set ofnucleic acids which is designed to specifically amplify the partial DNAregion containing the base at position 1018 of the glycoprotein Iba geneonly in the case where the base at position 1018 of the glycoprotein Ibagene in a nucleic acid sample is T.

In the above mention, kits are constructed by selecting two or morenucleic acids from the group consisting of (5) to (8). However, kits maybe constructed by making a group consisting of two or more nucleic acidsarbitrarily selected from (5) to (8) and selecting two or more sets ofnucleic acids from such a group. For example, kits may be constructed byselecting two or more nucleic acids from the group consisting of (5),(7) and (8) (nucleic acids for analyzing polymorphisms with threehighest odds ratio in Example mentioned below).

A kit for detecting the genotype, comprising two or more sets of nucleicacids selected from the group consisting of the following (1) to (4):

(1) a set of nucleic acids which is designed to specifically amplify thepartial DNA region containing the base at position 1648 of theglycoprotein Ia gene and which consists of a sense primer thatspecifically hybridizes the partial DNA region containing the base atposition 1648 of the glycoprotein Ia gene whose base at position 1648 isA and/or a sense primer that specifically hybridizes the partial DNAregion containing the base at position 1648 of the glycoprotein Ia genewhose gene at position 1648 is G and of an antisense primer thatspecifically hybridizes a partial region of the glycoprotein Ia gene;

(2) a set of nucleic acids which is designed to specifically amplify thepartial DNA region containing the base at position 190 of the chemokinereceptor 2 gene and which consists of an antisense primer thatspecifically hybridizes the partial DNA region containing the base atposition 190 of the chemokine receptor 2 gene whose base at position 190is G and/or an antisense primer that specifically hybridizes the partialDNA region containing the base at position 190 of the chemokine receptor2 gene whose gene at position 190 is A and of a sense primer thatspecifically hybridizes a partial region of the chemokine receptor 2gene;

(3) a set of nucleic acids which is designed to specifically amplify thepartial DNA region containing the base at position 1100 of theapolipoprotein C-III gene; and

(4) a set of nucleic acids which is designed to specifically amplify thepartial DNA region containing the base at position 825 of the G-proteinβ3 subunit gene and which consists of a sense primer that specificallyhybridizes the partial DNA region containing the base at position 825 ofthe G-protein β3 subunit gene whose base at position 825 is C and/or asense primer that specifically hybridizes the partial DNA regioncontaining the base at position 825 of the G-protein β3 subunit genewhose gene at position 825 is T and of an antisense primer thatspecifically hybridizes a partial region of the G-protein β3 subunitgene.

In the above mention, kits are constructed by selecting two or morenucleic acids from the group consisting of (1) to (4). However, kits maybe constructed by making a group consisting of two or more nucleic acidsarbitrarily selected from (1) to (4) and selecting two or more sets ofnucleic acids from such a group. For example, kits may be constructed byselecting two or more nucleic acids from the group consisting of (2) to(4) (nucleic acids for analyzing polymorphisms with three highest oddsratios in Example mentioned below).

A kit for detecting the genotype, comprising two or more sets of nucleicacids selected from the group consisting of the following (5) to (8):

(5) a set of nucleic acids which is designed to specifically amplify thepartial DNA region containing the base at position −850 of the tumornecrosis factor-α gene and which consists of an antisense primer thatspecifically hybridizes the partial DNA region containing the base atposition −850 of the tumor necrosis factor-α gene whose base at position−850 is C and/or an antisense primer that specifically hybridizes thepartial DNA region containing the base at position −850 of the tumornecrosis factor-α gene whose gene at position −850 is T and of a senseprimer that specifically hybridizes a partial region of the tumornecrosis factor-α gene;

(6) a set of nucleic acids which is designed to specifically amplify thepartial DNA region containing the base at position −238 of the tumornecrosis factor-α gene and which consists of an antisense primer thatspecifically hybridizes the partial DNA region containing the base atposition −238 of the tumor necrosis factor-α gene whose base at position−238 is G and/or an antisense primer that specifically hybridizes thepartial DNA region containing the base at position −238 of the tumornecrosis factor-α gene whose gene at position −238 is A and of a senseprimer that specifically hybridizes a partial region of the tumornecrosis factor-α gene;

(7) a set of nucleic acids which is designed to specifically amplify thepartial DNA region containing the base at position 3494 of the insulinreceptor substrate-1 gene and which consists of a sense primer thatspecifically hybridizes the partial DNA region containing the base atposition 3494 of the insulin receptor substrate-1 gene whose base atposition 3494 is G and/or a sense primer that specifically hybridizesthe partial DNA region containing the base at position 3494 of theinsulin receptor substrate-1 gene whose gene at position 3494 is A andof an antisense primer that specifically hybridizes a partial region ofthe insulin receptor substrate-1 gene; and

(8) a set of nucleic acids which is designed to specifically amplify thepartial DNA region containing the base at position 1018 of theglycoprotein Iba gene and which consists of a sense primer thatspecifically hybridizes the partial DNA region containing the base atposition 1018 of the glycoprotein Iba gene whose base at position 1018is C and/or a sense primer that specifically hybridizes the partial DNAregion containing the base at position 1018 of the glycoprotein Iba genewhose gene at position 1018 is T and of an antisense primer thatspecifically hybridizes a partial region of the glycoprotein Iba gene.

In the above mention, kits are constructed by selecting two or morenucleic acids from the group consisting of (5) to (8). However, kits maybe constructed by making a group consisting of two or more nucleic acidsarbitrarily selected from (5) to (8) and selecting two or more sets ofnucleic acids from such a group. For example, kits may be constructed byselecting two or more nucleic acids from the group consisting of (5),(7) to (8) (nucleic acids for analyzing polymorphisms with three highestodds ratio in Example mentioned below).

A kit for detecting the genotype comprising two or more sets of nucleicacids selected from the group consisting of the following (1) to (4);

(1) a set of nucleic acids which consists of a first nucleic acid thatspecifically hybridizes a partial region containing a base correspondingto the base at position 1648 in the antisense strand of the glycoproteinIa gene whose base at position 1648 is A and that is labeled with afirst labeling substance, of a second nucleic acid that specificallyhybridizes a partial region containing a base corresponding to the baseat position 1648 in the antisense strand of the glycoprotein Ia genewhose base at position 1648 is G and that is labeled with a secondlabeling substance, and of the third nucleic acid that specificallyhybridizes a partial region in the sense strand of the glycoprotein Iagene and that can specifically amplify the partial DNA region containingthe base at position 1648 of the glycoprotein Ia gene in concurrent usewith the above first or second nucleic acid;

(2) a set of nucleic acids which consists of a first nucleic acid thatspecifically hybridizes a partial region containing the base at position190 in the sense strand of the chemokine receptor 2 gene whose base atposition 190 is G and that is labeled with a first labeling substance,of a second nucleic acid that specifically hybridizes a partial regioncontaining the base at position 190 in the sense strand of the chemokinereceptor 2 gene whose base at position 190 is A and that is labeled witha second labeling substance, and of the third nucleic acid thatspecifically hybridizes a partial region in the antisense strand of thechemokine receptor 2 gene and that can specifically amplify the partialDNA region containing the base at position 190 of the chemokine receptor2 in concurrent use with the above first or second nucleic acid;

(3) a set of nucleic acids which consists of first and second nucleicacids that are designed to specifically amplify the partial DNA regioncontaining the base at position 1100 of the apolipoprotein C-III gene,of a third nucleic acid that specifically hybridizes the nucleic acidwhich is amplified by the use of the first and second nucleic acidsusing the apolipoprotein C-III gene in which the base at position 1100is C as a template and which is labeled with a first labeling substance,and of a fourth nucleic acid that specifically hybridizes a nucleic acidwhich is amplified by the use of the first and second nucleic acids byusing apolipoprotein C-III gene in which the base at position 1100 is Tas a template and which is labeled with a second labeling substance; and

(4) a set of nucleic acids which consists of a first nucleic acid thatspecifically hybridizes a partial region containing the base at position825 in the antisense strand of the G-protein 13 subunit gene whose baseat position 825 is C and that is labeled with a first labelingsubstance, of a second nucleic acid that specifically hybridizes apartial region containing the base at position 825 in the antisensestrand of the G-protein β3 subunit gene whose base at position 825 is Tand that is labeled with a second labeling substance, and of the thirdnucleic acid that specifically hybridizes a partial region in the sensestrand of the G-protein β3 subunit gene and that can specificallyhybridize the partial DNA region containing the base at position 825 ofthe G-protein β3 subunit gene in concurrent use with the above first orsecond nucleic acid.

In the above mention, kits are constructed by selecting two or morenucleic acids from the group consisting of (1) to (4). However, kits maybe constructed by making a group consisting of two or more nucleic acidsarbitrarily selected from (1) to (4) and selecting two or more nucleicacids from such a group. For example, kits may be constructed byselecting two or more nucleic acids from the group consisting of (2) to(4) (nucleic acids for analyzing polymorphisms with three highest oddsratios in Example mentioned below).

A kit for detecting the genotype comprising two or more sets of nucleicacids selected from the group consisting of the following (5) to (8);

(5) a set of nucleic acids which consists of a first nucleic acid thatspecifically hybridizes a partial region containing the base at position−850 in the sense strand of the tumor necrosis factor α gene whose baseat position −850 is C and that is labeled with a first labelingsubstance, of a second nucleic acid that specifically hybridizes apartial region containing the base at position −850 in the sense strandof the tumor necrosis factor α gene whose base at position −850 is T andthat is labeled with a second labeling substance, and of the thirdnucleic acid that specifically hybridizes a partial region in theantisense strand of the tumor necrosis factor α gene and that canspecifically amplify the partial DNA region containing the base atposition −850 of the tumor necrosis factor α in concurrent use with theabove first or second nucleic acid;

(6) a set of nucleic acids which consists of a first nucleic acid thatspecifically hybridizes a partial region containing the base at position−238 in the sense strand of the tumor necrosis factor α gene whose baseat position −238 is G and that is labeled with a first labelingsubstance, of a second nucleic acid that specifically hybridizes apartial region containing the base at position −238 in the sense strandof the tumor necrosis factor α gene whose base at position −238 is A andthat is labeled with a second labeling substance, and of the thirdnucleic acid that specifically hybridizes a partial region in theantisense strand of the tumor necrosis factor α gene and that canspecifically amplify the partial DNA region containing the base atposition −238 of the tumor necrosis factor α in concurrent use with theabove first or second nucleic acid;

(7) a set of nucleic acids which consists of a first nucleic acid thatspecifically hybridizes a partial region containing the base at position3494 in the antisense strand of the insulin receptor substrate-1 genewhose base at position 3494 is G and that is labeled with a firstlabeling substance, of a second nucleic acid that specificallyhybridizes a partial region containing the base at position 3494 in theantisense strand of the insulin receptor substrate-1 gene whose base atposition 3494 is A and that is labeled with a second labeling substance,and of the third nucleic acid that specifically hybridizes a partialregion in the sense strand of the insulin receptor substrate-1 gene andthat can specifically amplify the partial DNA region containing the baseat position 3494 of the insulin receptor substrate-1 gene in concurrentuse with the above first or second nucleic acid; and

(8) a set of nucleic acids which consists of a first nucleic acid thatspecifically hybridizes a partial region containing the base at position1018 in the antisense strand of the glycoprotein Iba gene whose base atposition 1018 is C and that is labeled with a first labeling substance,of a second nucleic acid that specifically hybridizes a partial regioncontaining the base at position 1018 in the antisense strand of theglycoprotein Iba gene whose base at position 1018 is T and that islabeled with a second labeling substance, and of the third nucleic acidthat specifically hybridizes a partial region in the sense strand of theglycoprotein Iba gene and that can specifically amplify the partial DNAregion containing the base at position 1018 of the glycoprotein Iba genein concurrent use with the above first or second nucleic acid.

In the above mention, kits are constructed by selecting two or morenucleic acids from the group consisting of (5) to (8). However, kits maybe constructed by making a group consisting of two or more nucleic acidsarbitrarily selected from (5) to (8) and selecting two or more nucleicacids from such a group. For example, kits may be constructed byselecting two or more nucleic acids from the group consisting of (5),(7) to (8) (nucleic acids for analyzing polymorphisms with three highestodds ratios in Example mentioned below).

In the above-mentioned kits, one or two or more of reagents (buffer,reagent for reaction, and reagent for detection, etc.) may be combinedin response to the usage of the kit.

The present invention is hereinafter described in more detail by way ofExamples.

EXAMPLE 1 Selection of Gene Polymorphism

By using several kinds of public databases including PubMed [NationalCenter for Biological Information (NCBI)], Online Mendelian inheritancein Men (NCBI), Single Nucleotide Polymorphism (NCBI), etc., from acomprehensive viewpoint including vascular biology, platelet-leukocytebiology, coagulation and fibrinolysis system, a metabolic factor such aslipid, sugar, etc., 71 genes which were estimated to be associated withcoronary arteriosclerosis, coronary artery spasm, hypertension, diabetesmellitus, hyperlipidemia, etc. were extracted from genes which had beenpreviously reported. Furthermore, among the polymorphisms existing inthese genes, 112 polymorphisms including polymorphisms which exist inpromoter regions or exons, or polymorphisms which were located in splicedonor sites or acceptor sites and expected to be associated with thefunctional changes of gene products were selected (FIGS. 1 and 2).

EXAMPLE 2 Determination of Gene Polymorphism

The study population comprised 1940 Japanese individuals (1107 men and833 women) who either visited outpatient clinics of or were admitted toone of the 15 participating hospitals between July 1994 and December2001. A total of 1067 subjects (574 men and 493 women) either hadhypertension [systolic blood pressure of ≧140 mmHg or diastolic bloodpressure of ≧90 mmHg, or both] or had taken antihypertensive drugs.Cases with coronary artery disease, valvular heart disease, congenitalmalformations of the heart or vessels, or renal or endocrinologicdiseases that cause secondary hypertension were excluded from the study.The 873 control subjects (533 men and 340 women) with normal bloodpressure (systolic blood pressure of <140 mmHg and diastolic bloodpressure of <90 mmHg) were recruited from individuals who were found tohave at least one of the conventional risk factors for coronary arterydisease, including habitual cigarette smoking (≧10 cigarettes daily),obesity [body mass index of >26 kg/m²], diabetes mellitus (fasting bloodglucose of >126 mg/dL or hemoglobin A_(1c) of >6.5%, or both),hypercholesterolemia (serum total cholesterol of >220 mg/dL), andhyperuricemia (serum uric acid of >7.7 mg/dL for men or >5.5 mg/dL forwomen), but who had no history of coronary artery disease. Thesecontrols showed normal resting electrocardiogram, and also in exercisetolerance test, no myocardial ischemic change was shown. Blood pressurewas measured with subjects in the seated position according to theguidelines of the American Heart Association (Perloff D, Grim C, FlackJ, Frohlich E D, Hill M, McDonald M, Morgenstern B Z. Human bloodpressure determination by sphygmomanometry. Circulation. 1993; 88:2640-2470.).

From each of the subjects, 7 mL of venous blood was collected in a tubecontaining 50 mmol/L EDTA-2Na and genome DNA was extracted by using aDNA extraction kit (Qiagen, Chatsworth, Calif.). Genotypes of singlenucleotide polymorphisms were determined with a fluorescence- orcolorimetry-based allele-specific primer-probe assay system (Toyobo GeneAnalysis, Tsuruga, Japan) (see FIG. 3). DNA fragment containing apolymorphism site was amplified by polymerase chain reaction (PCR) byusing two kinds of allele specific sense primers (or antisense primers)whose 5′ end were labeled with fluorescein isothiocyanate (FITC) orTexas red (TxR) and an antisense primer (or a sense primer) whose 5′ endwas labeled with biotin. Alternatively, DNA fragment containingpolymorphism site was amplified by PCR by using two kinds of allelespecific sense (or antisense) primers and an antisense (or a sense)primer whose 5′ end was labeled with biotin, or by using a sense primerand an antisense primer whose 5′ end was labeled with biotin. Thereaction solution (25 μL) contained 20 ng of DNA, 5 pmol of each primer,0.2 mmol/L of each deoxynucleoside triphosphate, 1 to 4 mmol/L of MgCl₂,1 U of DNA polymerase (rTaq or KODplus; Toyobo Co., Ltd. Osaka, Japan)in corresponding DNA polymerase buffer. The amplification protocolcomprised an initial denaturation at 95° C. for 5 minutes; 35 to 45cycles of denaturation at 95° C. for 30 seconds, annealing at 55 to67.5° C. for 30 seconds, extension at 72° C. for 30 seconds, and a finalextension at 72° C. for 2 minutes.

For determination of genotype by fluorescence, amplified DNA wasincubated with a solution containing streptavidin-conjugated magneticbeads in 96-well plates at room temperature. The plates were placed on amagnetic stand, supernatants were collected from the wells and thentransferred to the wells of a 96-well plate containing 0.01 M NaOH,followed by measuring fluorescence by microplate reader at excitationwavelength and fluorescence wavelength of 485 nm and 538 nm for FITC andat excitation wavelength and fluorescence wavelength of 584 nm and 612nm for TxR. Furthermore, for determination of genotype by colorimetry,amplified DNA was denatured with 0.3 M NaOH and then subjected tohybridization at 37° C. for 30 min in hybridization buffer containing 35to 40% formamide with any of allele-specific capture probes fixed to thebottom of the wells of a 96-well plate. After thorough washing of thewells, alkaline phosphatase-conjugated streptavidin was added to eachwell and the plate was shaken at 37° C. for 15 min. The wells were againwashed, and, after the addition of a solution containing 0.8 mM2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium(monosodium salt) and 0.4 mM 5-bromo-4-chloro-3-indolyl phosphatep-toluidine salt, the absorbance at 450 nm was measured.

To confirm the accuracy of genotyping by this method, DNA samples of 50people were selected at random, and the samples were subjected toPCR-restriction fragment length polymorphism method or direct sequencingmethod of PCR products. In any samples, the genotype determined by theallele specific primer-probe measurement system was identical to thatdetermined by PCR-restriction fragment length polymorphism method ordirect sequencing method.

Statistical analysis in the following association study was carried outas follows. Quantitative clinical data were compared between patientswith hypertension and controls by the unpaired Student's t test or theMann-Whitney U test. Qualitative data were compared by the chi-squaretest. Allele frequencies were estimated by the gene counting method, andthe chi-square test was used to identify significant departures fromHardy-Weinberg equilibrium. The present inventors performed multivariatelogistic regression analysis to adjust risk factors, with hypertensionas a dependent variable and independent variables including age, bodymass index (BMI), smoking status (0=nonsmoker, and 1=smoker), metabolicvariables (0=no history of diabetes mellitus, hypercholesterolemia, orhyperuricemia; and 1=positive history), and genotype of eachpolymorphism. Each genotype was assessed according to dominant,recessive, and additive genetic models, and the P value, odds ratio, and95% confidence interval were calculated. For combined genotype analyses,the present inventors performed the stepwise forward selection method ofmultivariate logistic regression to calculate odds ratios for eachcombined genotype.

EXAMPLE 3 Selection of Polymorphism Associated with Hypertension andDevelopment of Method for Diagnosing Hypertension

The present inventors performed an association study of the 112polymorphisms of the 71 candidate genes with myocardial infarction in451 men (myocardial infarction: 219, control: 232) and in 458 women(myocardial infarction: 226, control: 232) in the previous report(Yamada Y, Izawa H, Ichihara S, et al. Prediction of the risk ofmyocardial infarction from polymorphisms in candate genes. N Engl J Med.in press). In this study, the present inventors have found that 19 and18 single nucleotide polymorphisms were associated with the developmentof myocardial infarction in men and women, respectively, which includedcandidate genes of hypertension (see FIGS. 1, 2 and 4). In this Example,a large scale association study on the association of these singlenucleotide polymorphisms with hypertension was carried out in total 1940cases.

The background data of all 1940 participants (1107 men and 833 women)are shown in FIG. 5. For men, age, BMI, the prevalence of hyperuricemia,and the serum concentration of creatinine as well as systolic anddiastolic blood pressure were significantly greater, and the prevalenceof smoking was significantly lower, in subjects with hypertension thanin controls. For women, age, BMI, and the prevalence ofhypercholesterolemia and hyperuricemia as well as systolic and diastolicblood pressure were significantly greater in subjects with hypertensionthan in controls.

Multivariate logistic regression analysis with adjustment for age, BMI,and the prevalence of smoking, diabetes mellitus, hypercholesterolemia,and hyperuricemia revealed that 4 of the 19 polymorphisms examined formen and 4 of the 18 polymorphisms examined for women were significantlyassociated with hypertension (P<0.05 in either a dominant or recessivegenetic model) (see FIG. 6). The genotype distributions of thesepolymorphisms are shown (see FIG. 7).

The present inventors performed the stepwise forward selection method ofmultivariate logistic regression analysis (see FIG. 8) with either adominant or recessive model for each polymorphism based on the P value(the lower P value) for association with hypertension shown in FIG. 6.The chromosomal loci of the corresponding genes are also shown in FIG.8.

The −850C→T and −238G→A polymorphisms of the tumor necrosis factor-αgene were not in linkage disequilibrium [pairwise linkage disequilibriumcoefficient, D′(D/D_(max)), of −0.310 and standardized linkagedisequilibrium coefficient, r, of −0.020; P=0.613, chi-square test].Odds ratios for susceptibility to hypertension based on combinedgenotypes with the stepwise forward selection method for men and womenseparately are shown in FIGS. 9 and 11(A) and in FIGS. 10 and 11(B),respectively. For men, combined genotype analysis of the fourpolymorphisms (GPIa (1648A→G) polymorphism, CCR2 (190G→A) polymorphism,ApoC-III (1100C→T) polymorphism, GPβ3 (825C→T) polymorphism) revealedthat the maximal odds ratio was 5.34 (FIGS. 9 and 11(A)). For women,combined genotype analysis of the four polymorphisms (TNFa (−850C→T)polymorphism, TNFa (−238G→A) polymorphism, IRS-1 (3494G→A) polymorphism,GPIba (1018C→T) polymorphism) revealed that the maximal odds ratio was46.86 (FIGS. 10 and 11(B)).

As mentioned above, multivariate logistic regression analysis revealedthat four SNPs related to hypertension in men and women, respectively.That is to say, the relation of hypertension to 19 SNPs for men and 18SNPs for women was examined in a large-scale association study with 1940individuals, and four each of the polymorphism related to hypertensiondevelopment in men and women were identified. Furthermore, the presentinventors developed a genetic risk diagnosis system for hypertensionbased on the combined genotypes for these SNPs that yielded maximal oddsratios of 5.34 for men and 46.86 for women by the stepwise forwardselection method of multivariate logistic regression analysis.

The regulation of blood pressure involves both the integration of avariety of biological systems that control the structure and tone of thevasculature and the volume and composition of body fluid, as well as theadaptation of these systems to constantly changing physiological needs(Laouel J-M, Rohrwasser A. Development of genetic hypotheses inessential hypertension. J Hum Genet. 2001; 46: 299-306). The relation ofhypertension to 19 SNPs for men and 18 SNPs for women examined for menand women, respectively, in the present study were selected on the basisof a comprehensive overview of vascular biology, platelet-leukocytebiology, the fibrinolysis system, as well as lipid and glucosemetabolism and other metabolic factors. Actually, the genes now shown tobe associated with hypertension may play roles in diverse aspects of theetiology of this condition, including vascular biology (G protein β3subunit), inflammation (tumor necrosis factor-α), monocyte andlymphocyte biology (chemokine receptor 2), platelet function(glycoproteins Ia and Iba), lipid metabolism (apolipoprotein C-III), andinsulin and glucose metabolism (insulin receptor substrate-1). Themaximal odds ratios obtained with the genetic risk diagnosis system forhypertension developed by the present inventors (5.34 for men and 46.86for women) appear to be the highest such values reported by large-scaleassociation studies of hypertension particularly in women. Among theeight polymorphisms associated with hypertension, the −850C→T and−238G→A polymorphisms of the tumor necrosis factor-α gene yielded thehighest odds ratio for predisposition to hypertension in women. Thetumor necrosis factor-α gene locus was previously shown to be associatedwith obesity-related hypertension in French Canadian (Pausova Z,Deslauriers B, Gaudet D, Tremblay J, Kotchen T A, Larochelle P, Cowley AW, Hamet P. Role of tumor necrosis factor-α gene locus in obesity andobesity-associated hypertension in French Canadians. Hypertension. 2000;36: 14-19). Furthermore, a −308A→G polymorphism of the tumor necrosisfactor-α gene also previously showed a tendency to associate withessential hypertension, although statistical significance was notachieved (Frossard P M, Gupta A, Pravica V, Perry C, Hutchinson I V,Lukic M L. A study of five human cytokine genes in human essentialhypertension. Mol Immunol. 2002; 38: 969-976.). The serum concentrationof this the tumor necrosis factor-α was associated with systolic bloodpressure and insulin resistance in a native Canadian population (ZinmanB, Hanley A J G, Harris S B, Kwan J, Fantus I G. Circulating tumornecrosis factor-α concentrations in a native Canadian population withhigh rates of type 2 diabetes mellitus. J Clin Endocrinol Metab. 1999;84: 272-278.). Tumor necrosis factors stimulates the production of thepotent vasoconstrictor endothelin-1 (Kahaleh M B, Fan P S. Effect ofcytokines on the production of endothelin by endothelial cells. Clin ExpRheumatol. 1997; 15: 163-167.) and the serum concentrations of these twoagents were positively correlated in subjects with obesity (Winkler G,Lakatos P, Salamon F, Nagy Z, Speer Q Kovacs M, Harmos G, Dworak O, CsehK. Elevated serum TNF-a level as a link between endothelial dysfunctionand insulin resistance in normotensive obese patients. Diabetes Med.1999; 16: 207-211.). These findings and the above-mentioned results bythe present inventors suggest that the tumor necrosis factor-α gene is acandidate locus for susceptibility to hypertension. With regard to theother six polymorphisms associated with hypertension in the presentstudy, the 825C→T polymorphism of the G protein β3 subunit gene waspreviously associated with hypertension (Siffert W, Rosskop D, SiffertG, Busch S, Moritz A, Erbel R, Sharma A M, Ritz E, Wichmann H-E, JakobsK H, Horsthemke B. Association of a human G-protein 13 subunit variantwith hypertension. Nat Genet. 1998; 18: 45-48.), and a polymorphism ofthe apolipoprotein C-III gene was also reported to be associated withblood pressure (Tas S, Abdella N A. Blood pressure, coronary arterydisease, and glycaemic control in type 2 diabetes mellitus: relation toapolipoprotein-CIII gene polymorphism. Lancet. 1994; 343: 1994-1995.).Chemokine receptor 2 and insulin receptor substrate-1 genes have beenshown to contribute to the development of hypertension (Bush E, Maeda N,Kuziel W A, Dawson T C, Wilcox J N, DeLeon H, Taylor W R. CC chemokinereceptor 2 is required for macrophage infiltration and vascularhypertrophy in angiotensin II-induced hypertension. Hypertension. 2000;36: 360-363., Abe H, Yamada N, Kamata K, Kuwaki T, Shimada M, Osuga J,Shionoiri F, Yahagi N, Kadowaki T, Tamemoto H, Ishibashi S, Yazaki Y,Makuuchi M. Hypertension, hypertriglyceridemia, and impairedendothelium-dependent vascular relaxation in mice lacking insulinreceptor substrate-1. J Clin Invest. 1998; 101: 1784-1788.).Furthermore, platelet activation may be important in the etiology ofessential hypertension (Andrioli G, Ortolani R, Fontana L, Gaino S,Bellavite P, Lechi C, Minuz P, Manzato F, Tridente G, Lechi A. Study ofplatelet adhesion in patients with uncomplicated hypertension. JHypertens. 1996; 14: 1215-1221., Dockrell M E, Walker B R, Noon J P,Watt G C, Williams B C, Webb D J. Platelet aggregation in young mencontrasting predisposition to high blood pressure. Am J Hypertens. 1999;12: 115-119., Bereczki C, Tur S, Nemeth I, Sallai E, Torday C, Nagy E,Haszon I, Papp F. The roles of platelet function, thromboxane, bloodlipids, and nitric oxide in hypertension of children and adolescents.Prostaglandins Leukot Essent Fatty Acids. 2000; 62: 293-297.); althoughthe polymorphisms in the glycoprotein Ia and glycoprotein Iba genesstudied here have been associated with coronary artery disease (Kroll H,Gardemann A, Fechter A, Haberbosch W, Santoso S. The impact of theglycoprotein Ia collagen receptor subunit A1648G gene polymorphism oncoronary artery disease and acute myocardial infarction. Thromb Haemost.2000; 83: 392-396., Murata M, Matsubara Y, Kawano K, et al. Coronaryartery disease and polymorphisms in a receptor mediating shearstress-dependent platelet activation. Circulation. 1997; 96: 3281-6.),they have not previously been associated with hypertension.

It is possible that some of the polymorphisms examined in the presentExamples are in linkage disequilibrium with polymorphisms of othernearby genes that are actually responsible for the development ofhypertension. The present results indicate, however, that glycoproteinIa, chemokine receptor 2, apolipoprotein C-III, and the G protein β3subunit are susceptibility loci for hypertension in Japanese men, andthat tumor necrosis factor-α, insulin receptor substrate-1, andglycoprotein Iba constitute such loci in Japanese women. Moreover, thecombined genotypes for these polymorphisms may prove informative fordetermination of the genetic risk for hypertension. The geneticdiagnosis system by the present inventors should therefore contribute tothe primary prevention of hypertension and of cardiovascular diseases,stroke, or renal diseases induced by this condition.

The present invention is not limited to the description of the aboveembodiments. A variety of modifications, which are within the scopes ofthe following claims and which are achieved easily by a person skilledin the art, are included in the present invention.

INDUSTRIAL APPLICABILITY

According to the present invention, gene polymorphisms associated withhypertension are analyzed and the genotype in a nucleic acid sample isdetected. By using the information about the polymorphisms obtained bythe detection of the genotype, diagnosis of the risk for hypertensionwith high accuracy and high predictability can be carried out.Therefore, the present invention provides an effective means forunderstanding in advance the risk of development of hypertension, sothat it can be expected that the means should therefore contribute tothe primary prevention of hypertension as well as to contribute theprevention of cardiovascular diseases, stroke, renal diseases, or thelike, induced by hypertension. Furthermore, according to the presentinvention, auxiliary information useful for the diagnosis ofhypertension can be obtained so as to enable an appropriate treatmentand therefore prognosis can be improved. Furthermore, since the presentinvention provides useful information in elucidating the developmentmechanism of hypertension, it also provides an extremely important meansfor establishing a preventing method for hypertension.

1. A method for detecting the genotype in a nucleic acid sample, the method comprising the following step (a): (a) analyzing two or more polymorphisms selected from the group consisting of the following (1) to (4) in a nucleic acid sample: (1) a polymorphism at the base number position 1648 of the glycoprotein Ia gene; (2) a polymorphism at the base number position 190 of the chemokine receptor 2 gene; (3) a polymorphism at the base number position 1100 of the apolipoprotein C-III gene; and (4) a polymorphism at the base number position 825 of G-protein β3 subunit gene.
 2. A method for detecting the genotype in a nucleic acid sample, the method comprising the following step (b): (b) analyzing two or more polymorphisms selected from the group consisting of the following (5) to (8) in a nucleic acid sample: (5) a polymorphism at the base number position −850 of the tumor necrosis factor-α gene; (6) a polymorphism at the base number position −238 of the tumor necrosis factor-α gene; (7) a polymorphism at the base number position 3494 of the insulin receptor substrate-1 gene; and (8) a polymorphism at the base number position 1018 of the glycoprotein Iba gene.
 3. A method for diagnosing the risk for hypertension, the method comprising the following steps (i) to (iii): (i) analyzing two or more polymorphisms selected from the group consisting of the following (1) to (4) in a nucleic acid sample: (1) a polymorphism at the base number position 1648 of the glycoprotein Ia gene; (2) a polymorphism at the base number position 190 of the chemokine receptor 2 gene; (3) a polymorphism at the base number position 1100 of the apolipoprotein C-III gene; and (4) a polymorphism at the base number position 825 of G-protein β3 subunit gene. (ii) determining, based on the information about polymorphism which was obtained in the step (i), the genotype in the nucleic acid sample; and (iii) assessing, based on the genotype determined, a genetic risk for hypertension.
 4. A method for diagnosing the risk for hypertension, the method comprising the following steps (iv) to (vi): (iv) analyzing two or more polymorphisms selected from the group consisting of the following (5) to (8) in a nucleic acid sample: (5) a polymorphism at the base number position −850 of the tumor necrosis factor-α gene; (6) a polymorphism at the base number position −238 of the tumor necrosis factor-α gene; (7) a polymorphism at the base number position 3494 of the insulin receptor substrate-1 gene; and (8) a polymorphism at the base number position 1018 of the glycoprotein Iba gene. (v) determining, based on the information about polymorphism which was obtained in the step (iv), the genotype in the nucleic acid sample; and (vi) assessing, based on the genotype determined, a genetic risk for hypertension.
 5. A kit for detecting the genotype, comprising two or more of nucleic acids selected from the group consisting of the following (1) to (4): (1) a nucleic acid for analyzing a polymorphism at the base number position 1648 of the glycoprotein Ia gene; (2) a nucleic acid for analyzing a polymorphism at the base number position 190 of the chemokine receptor 2 gene; (3) a nucleic acid for analyzing a polymorphism at the base number position 1100 of the apolipoprotein C-III gene; and (4) a nucleic acid for analyzing a polymorphism at the base number position 825 of G-protein β3 subunit gene.
 6. A kit for detecting the genotype, comprising two or more of nucleic acids selected from the group consisting of the following (5) to (8): (5) a nucleic acid for analyzing a polymorphism at the base number position −850 of the tumor necrosis factor-α gene; (6) a nucleic acid for analyzing a polymorphism at the base number position −238 of the tumor necrosis factor-α gene; (7) a nucleic acid for analyzing a polymorphism at the base number position 3494 of the insulin receptor substrate-1 gene; and (8) a nucleic acid for analyzing a polymorphism at the base number position 1018 of the glycoprotein Iba gene.
 7. Fixed nucleic acids comprising the following two or more nucleic acids selected from the group consisting of the following (1) to (4) fixed to an insoluble support: (1) a nucleic acid for analyzing a polymorphism at the base number position 1648 of the glycoprotein Ia gene; (2) a nucleic acid for analyzing a polymorphism at the base number position 190 of the chemokine receptor 2 gene; (3) a nucleic acid for analyzing a polymorphism at the base number position 1100 of the apolipoprotein C-III gene; and (4) a nucleic acid for analyzing a polymorphism at the base number position 825 of G-protein β3 subunit gene.
 8. Fixed nucleic acids comprising the following two or more nucleic acids selected from the group consisting of the following (5) to (8) fixed to an insoluble support: (5) a nucleic acid for analyzing a polymorphism at the base number position −850 of the tumor necrosis factor-α gene; (6) a nucleic acid for analyzing a polymorphism at the base number position −238 of the tumor necrosis factor-α gene; (7) a nucleic acid for analyzing a polymorphism at the base number position 3494 of the insulin receptor substrate-1 gene; and (8) a nucleic acid for analyzing a polymorphism at the base number position 1018 of the glycoprotein Iba gene. 